Metabolic functions and inheritance of the microsporidian mitosome

Phd ThesisMicrosporidia are a group of obligate intracellular parasites of economic and medical importance. Many aspects of microsporidian genomes and cell biology are a result of an extensive reductive evolution during adaptation to their intracellular parasitic lifestyle. Microsporidian mitochondrial homologues called mitosomes have only a single known conserved metabolic function in biosynthesis of the essential iron-sulfur clusters. Based on genomic data an additional function of the mitosome in the alternative respiratory pathway (ARP) was proposed for some microsporidians including human pathogenic Trachipleistophora hominis. This thesis provides the first direct experimental evidence for a mitosomal localization of the two components of the ARP in T. hominis. Quantitative analyses of the immunofluorescence data together with western blotting experiments provided results consistent with the life cycle-stage specific function of the organelle. In the proliferative stages of the T. hominis life cycle, capable of stealing ATP from the host, mitosomes seem to function mostly in the biosynthesis of the essential iron sulfur clusters. The ARP proteins are enriched in the T. hominis spores, which is consistent with the hypothetical functions of the mitosome in energy metabolism of the spore that is unable to rely on its host for ATP production. This thesis also provides the first bioinformatics characterization of the molecular machineries involved in the processes required for inheritance of the microsporidian mitosomes: organelle fission and segregation during the cell division. Specific antibodies were generated and used to detect the microsporidian spindle pole body (SPB), an organelle hypothesized to play a role in inheritance of the microsporidian mitosomes. Double labelling experiments using the specific antibodies against the SPB and mitosomal markers provide evidence for a stable connection between the two organelles throughout the life cycle of the parasite.European Union as a part of a Marie Curie Initial Training Network Symbiomics

Comparing the rate of growth and metabolic efficiency of yeast experiencing environmental stress or genetic damage

Physical stresses, toxic substances, and mutations can cause marked decline in the rate of growth (RG). We report that the efficiency of growth (EG), i.e. converting glucose into biomass, responds less profoundly. It remains nearly unaffected for some physical and chemical stresses, but decreases substantially for others, specifically those affecting membrane integrity or ion homeostasis. Mutations (gene deletions) can heavily reduce RG, but much less EG. Moreover, there is no apparent relation between the function of deleted gene and EG. Generally, assays of EG appear as more laborious, less precise, and less informative than those of RG

Biogenesis, inheritance and 3D ultrastructure of the microsporidian mitosome

Funding: This work was supported by a European Research Council Advanced Investigator Grant (ERC-2010-AdG-268701) to T.M.E., and a Wellcome Trust Programme Grant (Number 045404) to T.M.E. and J.M.L.During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2–12/cell) and Trachipleistophora hominis (14–18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome–mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).Publisher PDFPeer reviewe

Morphology and Phylogeny of the Ciliate Psilotricha silvicola n. sp. (Alveolata, Ciliophora) from Woodland Soils in the United Kingdom.

The genus Psilotricha was established by Stein in 1859, with P. acuminata as the type species within the family Oxytrichidae. This species lacked a full description until it was re-discovered in 2001, showing that its morphological and morphogenetic characters confirmed the inclusion in the family Oxytrichidae. Since then, the genus Psilotricha has had a convoluted taxonomy despite the morphological evidence available. In this paper, we describe a new Psilotricha species, Psilotricha silvicola n. sp., from woodland soils in Southern England (United Kingdom). The morphology was investigated in live and protargol-impregnated specimens. Our findings show that P. silvicola n. sp. shares morphological characteristics with P. acuminata, including the distinctive cell shape and the long and sparse cirri. Phylogenetic analysis of the 18S rRNA gene places this new species within the family Oxytrichidae, nested apart from the family Psilotrichidae (which includes the genera Urospinula, Psilotrichides and Hemiholosticha), in a clade containing species of the family Oxytrichidae. Furthermore, the morphology of another Psilotricha species, P. viridis, found in a freshwater pond in the same woodland area, is also here described, bringing additional insight into the taxonomy of the genus. Our findings provide further evidence for inclusion of the genus Psilotricha within the oxytrichids

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells

Mitosomes are highly reduced forms of mitochondria which have lost two of the ‘defining’ features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite’s life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells. Author summary Microsporidia are increasingly appreciated as ubiquitous pathogens with ecological, veterinary and medical importance. Here we provide detailed protocol for establishing a synchronised infection of microsporidian parasites T. hominis and E. cuniculi inside host RK13 cell lines. We demonstrate how this protocol in combination with molecular and cell biology techniques can be utilised to test the effects of anti-microsporidian drugs, and specific protein inhibitors on the parasite’s cell biology and infection cycle progression. Using these techniques with bespoke specific antibodies against T. hominis alternative respiration (AR) components—alternative oxidase (AOX) and glycerol-3-phosphate dehydrogenase (mtG3PDH)—we demonstrate their life cycle stage-dependent mitosomal localisation, and in combination with the specific AOX-inhibitor colletochlorin B we show an essential function of the mitosomal AOX during an early phase of the T. hominis infection cycle. Using bioinformatics and specific inhibitors we show that the role of the microsporidian minimal mitochondria in AR was subsequently lost in the course of further reductive evolution in some lineages including that of E. cuniculi. Additional RNAseq across T. hominis life cycle and experiments using antibodies against the mitosomal iron-sulphur clusters biosynthesis (ISC) protein NFS indicated that unlike the phylogenetically-restricted AR, the conserved ISC function of the mitosome is active during T. hominis proliferation

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells

An evolutionary path to altered cofactor specificity in a metalloenzyme

Place: London Publisher: Nature Publishing Group WOS:000542983700007Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity

Ciliophora sp. Raw sequence reads

Investigating the repeated convergent evolution of ciliate hydrogenosomes from three distinct lineages, using a combined genomic and transcriptomic dataset

Testing the effects of late addition of the coletochlorin B to the culture of RK13 cells infected with <i>T</i>. <i>hominis</i>.

(A) Images of representative T. hominis cells observed in the samples collected across the time course of infection where, colletochlorin B (CCB) was added to the medium 22 hours after the addition of spores to the non-infected host cells, when AOX and mtG3PDH labelled mitosomes were absent from virtually all parasite cells (S5 Fig). Representative cells of the parasite life cycle stages observed at each time point; meronts (54 hpi), sporonts (64–80 hpi), sporoblasts (64–120 hpi), and spores (80–130 hpi); were indicated on the images (white arrows). Time points displayed above the images refer to the time points after the addition of the spores. (B) Low magnification image of the CCB treated samples at 136 hpi. All infected host cells contained spore bags (white arrows), but no new infections were observed. Cells labelled with antibodies represent late meront stages from the initial infection that have not entered the spore formation stage of life cycle. All samples were double-labelled with the antibodies against ThNTT4 (red), and ThmtHSP70 (green). (TIFF)</p